https://repository.kulib.kyoto-u.ac.jp/dspace/bitstream/2433/49782/1/Yamanaka_Cell_131_5.pdf

Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors

If it were possible to reprogram differentiated human somatic cells into a pluripotent state, patient-specific and disease-specific stem cells could be developed. Previous work generated induced pluripotent stem (iPS) cells capable of germline transmission from murine somatic cells by transd

/pdf/induction-of-pluripotent-stem-cells-from-adult-human-3jc4cxk5x5.pdf

Induction of Pluripotent Stem Cells From Adult Human Fibroblasts by Defined Factors

Cell fate during development is defined by transcription factors that act as molecular switches to activate or repress specific gene expression programmes. The POU transcription factor Oct-3/4 (encoded by Pou5f1) is a candidate regulator in pluripotent and germline cells and is essential for the initial formation of a pluripotent founder cell population in the mammalian embryo. Here we use conditional expression and repression in embryonic stem (ES) cells to determine requirements for Oct-3/4 in the maintenance of developmental potency. Although transcriptional determination has usually been considered as a binary on-off control system, we found that the precise level of Oct-3/4 governs three distinct fates of ES cells. A less than twofold increase in expression causes differentiation into primitive endoderm and mesoderm. In contrast, repression of Oct-3/4 induces loss of pluripotency and dedifferentiation to trophectoderm. Thus a critical amount of Oct-3/4 is required to sustain stem-cell self-renewal, and up- or downregulation induce divergent developmental programmes. Our findings establish a role for Oct-3/4 as a master regulator of pluripotency that controls lineage commitment and illustrate the sophistication of critical transcriptional regulators and the consequent importance of quantitative analyses.

Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells.

http://web.mit.edu/7.31/restricted/pdfs/F05_and_earlier/Nichols.pdf

Formation of Pluripotent Stem Cells in the Mammalian Embryo Depends on the POU Transcription Factor Oct4

Functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cells

Various extrinsic markers have been used to label single cells in the early mouse embryo. However, they are appropriate only for short-term experiments because of their susceptibility to dilution. Studies on cell lineage and commitments have therefore depended mainly on exploiting genes as markers by combining cells from embryos that differ in genotype at particular loci. Tissue recombination and transplantation experiments using such indelible intrinsic markers have enabled the fate of different cell populations in the blastocyst to be determined with reasonable precision. The trophectoderm and inner cell mass (i.c.m.) give rise to distinct complementary groups of tissues in the later conceptus, as do the primitive endodermal and primitive ectodermal components of the more mature i.c.m. When cloned by blastocyst injection, single i.c.m. cells colonize only those parts of host conceptuses that are derived from their tissue of origin. Thus, while clonal descendants of early i.c.m. cells can contribute to all tissues other than those of trophectodermal origin, primitive endodermal and primitive ectodermal clones are restricted, respectively, to the extraembryonic endoderm versus all i.c.m. derivatives except the extraembryonic endoderm. Interestingly, individual primitive ectoderm cells can include both germ cells and somatic cells among their mitotic descendants. By using the genetically determined presence versus absence of cytoplasmic malic enzyme activity as a cell marker, the deployment of clones has been made visible in situ in whole-mount preparations of extraembryonic membranes. Very little mixing of donor and host cells was seen in either the endoderm of the visceral yolk sac or the mesodermal and ectodermal layers of the amnion. In contrast, mosaicism in the parietal endoderm was so fine grained that, in all except 1 of 15 fields from several specimens that were analysed, the arrangement of donor and host cells did not differ significantly from that expected on the basis of their random association.

Clonal analysis of early mammalian development.

Conditions were found for staining whole mid-gestation capsular parietal endoderms and visceral yolk sacs for malic enzyme activity that gave excellent discrimination between wildtype ( Mod-1 + /Mod-1 + ) cells and mutant ( Mod-l n /Mod-1 n ) cells that lack the cytoplasmic form of the enzyme. Reciprocal blastocyst injection experiments were undertaken in which single primitive endoderm cells of one genotype were transplanted into embryos of the other genotype. In addition, Mod-1 + /Mod-1 + early inner cell mass (ICM) cells were injected into Mod-1 n /Mod-1 n blastocysts, either in groups of two or three singletons or as daughter cell pairs. A substantial proportion of the resulting conceptuses showed mosaic histochemical staining in the parietal endoderm, visceral yolk sac, or in both these membranes. Stained cells were invariably intimately intermixed with unstained cells in the mosaic parietal endoderms. In contrast, one or both of two distinct patterns of staining could be discerned in mosaic visceral yolk sacs. The first, a conspicuously ‘coherent’ pattern, was found to be due to endodermal chimaerism; the second, a more diffuse pattern, was attributable to chimaerism in the mesodermal layer of this membrane. The overall distribution of cells with donor staining characteristics resulting from primitive endoderm versus early ICM cell injections was consistent with findings in earlier experiments in which allozymes of glucosephosphate isomerase were used as markers. The conspicuous lack of phenotypically intermediate cells in predominantly stained areas of mosaic membranes suggested that the histochemical difference between Mod-1 + /Mod-1 + and Mod-1 n /Mod-l n genotypes was cell-autonomous. This conclusion was strengthened by the results of staining mixed in vitro cultures of parietal endoderm in which presence or absence of phagocytosed melanin granules was used as an independent means of distinguishing wild type from null cells. By substituting tetranitro blue tetrazolium for nitro blue tetrazolium in the incubation medium, satisfactory differential staining was obtained for both the extraembryonic endoderm and other tissues of earlier postimplantation wild type versus null embryos. Finally, absence of cytoplasmic malic enzyme activity does not appear to have a significant effect on the viability or behaviour of mutant cells.

An in situ cell marker for clonal analysis of development of the extraembryonic endoderm in the mouse

BACKGROUND: Mammalian conceptuses typically have an approximately regular tetrahedral shape at the 4-cell stage. In the rabbit, this has been attributed to both 2-cell blastomeres dividing meridionally, but with the animal– vegetal axis of the second blastomere to divide rotating through roughly 90° before or during cytokinesis. The aim of the present study was to ascertain whether this was also true for the mouse. METHODS AND RESULTS: First, the distribution in regular tetrahedral 4-cell conceptuses of fluorescent microspheres applied to the vegetal polar region of one or both blastomeres at the 2-cell stage was analysed. Second, the ability of 2-cell stages to form regular tetrahedral 4-cell conceptuses after the previtelline space had been gelated to prevent blastomeres from rotating was also investigated. Neither experiment yielded evidence supporting blastomere rotation during second cleavage. Rather, the findings were consistent with the regular tetrahedral form of 4-cell conceptus resulting from meridional division of one blastomere and approximately equatorial division of the other. CONCLUSIONS: Second cleavage in the mouse typically yields 4-cell conceptuses with three distinct types of blastomere. While both products of the meridional division include all axial levels of the zygote, those of the equatorial division acquire only its vegetal or animal half.

/pdf/experimental-analysis-of-second-cleavage-in-the-mouse-3nuqjn30k1.pdf

Experimental analysis of second cleavage in the mouse

Culture of postimplantation conceptuses was used in conjunction with microsurgery to investigate the timing, the mechanism and the developmental regulation of chorioallantoic fusion in the mouse. The timing of fusion was determined in both freshly recovered conceptuses and in those that had been cultured from as early as the mid-streak stage. Attachment of the allantois to the chorion was found to have occurred in most conceptuses by the 6-somite stage, irrespective of whether they had been cultured. In investigating the mechanism of fusion, we wished to determine whether it depended on directed growth of the allantoic bud or on its differential adhesion to the chorion. Microsurgery was used to transplant allantoic tissue into the exocoelomic cavity of conceptuses from which the resident allantois had been removed. In synchronous grafting experiments, transplanted allantoises typically attached to the chorion despite loss of their connection with the hindgut region of the fetus. Hence selective attachment of the allantois to the chorion clearly cannot depend simply on its directed growth. While the transplanted allantoic tissue attached to the chorion selectively, it did not attach to it precociously, despite being favourably positioned to do so. These findings argue that the initial attachment of the allantois to the chorion depends on a selective adhesive mechanism that is developmentally regulated. Further grafting experiments in which donor conceptuses were either more or less advanced than hosts revealed that attachment of the allantois to the chorion depends primarily on the stage of the allantois rather than on the stage of the chorion. Collectively, these findings support the hypothesis that the initial stage of chorioallantoic fusion depends on selective adhesion between regionally differentiated mesodermal surfaces which is governed principally by the stage of development of the allantois.

Richard L. Gardner

Papers

Clonal analysis of early mammalian development.

An in situ cell marker for clonal analysis of development of the extraembryonic endoderm in the mouse

Experimental analysis of second cleavage in the mouse

An investigation into early placental ontogeny: allantoic attachment to the chorion is selective and developmentally regulated

A structurally defined mini-chromosome vector for the mouse germ line